Homologous recombination occurs in all cells and provides a major vehicle for creating new genetic information. Its pathways are closely related to those of DNA repair and mutagenesis. Only in E. coli, have the proteins that direct homologous recombination been purified and examined in detail. In vitro studies with these proteins, the RecA and SSB proteins, have focused upon and defined the substrates and end-products, but the intermediates and pathways of homologous recombination are largely unknown. This is the focus of this proposal. It is known that these reactions occur within large filamentous recombination structures produced by the helical arrangement of RecA protein about DNA. It is now imperative that the architecture of the recombination structure be elucidated and correlated with the known DNA template requirements and end-products of these reactions. The approach used here integrates advanced electron microscopy (EM) with other physical and biochemical methods. New methods for preserving ultrastructure of DNA-protein complexes for EM will be developed and applied. In the cell, recombination may be controlled by regulating the assembly of RecA protein onto single stranded DNA. This hypothesis will be tested. Additional questions to be answered include: How do the RecA protein-DNA filaments direct a search for homology? How rapidly does RecA protein turnover as strand exchange progresses? Can excess RecA protein change certain DNA repair pathways into recombinational pathways? What is the ultrastructure of plectonemic and paranemic joints in the recombination structure? We recently discovered that RecA protein can bring broken DNA ends into juxtaposition. This will be investigated in relation to strand exchange mechanisms to examine its possible role in DNA repair. The uvsX protein of bacteriophage T4 appears analagous to RecA protein, and parallel studies with it will be initiated in a effort to begin to establish the rules of homologous recombination common to all cells.